Hearing loss can cause great frustration amongst its sufferers. In some cases, these individuals contend with feelings of loneliness and depression because they find it difficult to communicate with the people around them. Currently, the ability to treat hearing loss relies heavily on the use of hearing aids; however, these devices do not restore hearing acuity to standard levels. This proposal focuses on creating and investigating potential therapeutic tools that could restore acuity and detection of sound to people suffering from hearing loss caused by a lack or loss of hair cells. The supporting cells located beneath the hair cells in sensory organs of the inner ear offer an excellent cellular source to target for hair cell regeneration. Hair cells and supporting cells are created from the same progenitor pool during development and in birds, hair cell loss stimulates supporting cells to divide and regenerate additional hair cells. The factors underlying the fate choice of supporting cell vs. hair cell may provide potential reagents for treating deafness and balance disorders. Previous research has focused on supplying adult supporting cells with the pro-hair cell transcription factor, Atoh1, to force those cells to switch to a hair cell fate, but an alternative approach is to identify and supply anti-supporting cell factors to assist in converting supporting cells to a new fate. Presently, members of the miRNA-183 family are likely candidates to serve as anti-supporting cell signals. Another miRNA, miR-9, may also prove to be influential in discouraging a supporting cell fate by decreasing the levels of a transcription factor, HES-1, that normally negatively regulates the pro-hair gene, Atoh1. The goals of this project are to investigate the role and mechanism by which miR-9 and the miR-183 family work during ear development as well as to construct new gene therapy tools encoding both the pro-hair cell factor (Atoh1) and presumed anti-supporting cell factors (miRNAs). Aim 1 will focus on the construction of a dual expression RCAS vector for Atoh1 and the miR-183 family or miR-9, along with an alternate version that encodes only the miRNAs. Aim 2 will involve delivery of these vectors into a chicken otocyst to observe their effects on hair cell development. With this proposal, we hope to elucidate the role of the 183 family of miRNAs and miR-9 in hair cell development along with one of its possible targets, HES-1, and create tools to generate new hair cells that may be used to treat cases of human deafness in the future.